High-throughput screening and bioinformatic analysis to ascertain compounds that prevent saturated fatty acid-induced β-cell apoptosis

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient Escherichia coli host strain and report an HTS approach based on colorimetric detection of H2O2-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H2O2 as co-substrate.

Download Full-text

Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110372257 ◽

2010 ◽

Vol 15 (6) ◽

pp. 663-670 ◽

Cited By ~ 20

Author(s):

Alice Kiselyuk ◽

Suzette Farber-Katz ◽

Tom Cohen ◽

Seung-Hee Lee ◽

Ifat Geron ◽

...

Keyword(s):

Cell Line ◽

Human Insulin ◽

High Throughput ◽

High Throughput Screening ◽

Promoter Activity ◽

Central Element ◽

Bhlh Transcription Factor ◽

Β Cell ◽

Insulin Promoter ◽

Short Term Exposure

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.

Download Full-text

High-throughput screening for fatty acid uptake inhibitors in humanized yeast identifies atypical antipsychotic drugs that cause dyslipidemias

The Journal of Lipid Research ◽

10.1194/jlr.d700015-jlr200 ◽

2007 ◽

Vol 49 (1) ◽

pp. 230-244 ◽

Cited By ~ 24

Author(s):

Hong Li ◽

Paul N. Black ◽

Aalap Chokshi ◽

Angel Sandoval-Alvarez ◽

Ravi Vatsyayan ◽

...

Keyword(s):

Fatty Acid ◽

Atypical Antipsychotic ◽

High Throughput ◽

High Throughput Screening ◽

Antipsychotic Drugs ◽

Fatty Acid Uptake ◽

Acid Uptake ◽

Atypical Antipsychotic Drugs ◽

Uptake Inhibitors

Download Full-text

A simple, reliable method for high-throughput screening for diabetes drugs using 3D β-cell spheroids

Journal of Pharmacological and Toxicological Methods ◽

10.1016/j.vascn.2016.08.005 ◽

2016 ◽

Vol 82 ◽

pp. 83-89 ◽

Cited By ~ 5

Author(s):

Jesal Amin ◽

Karthik Ramachandran ◽

S. Janette Williams ◽

Annie Lee ◽

Lesya Novikova ◽

...

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Reliable Method ◽

Β Cell ◽

Cell Spheroids

Download Full-text

Development of high-throughput screening assays for profiling snake venom Phospholipase A2 activity after high-resolution chromatographic fractionation

10.1101/2020.01.20.912758 ◽

2020 ◽

Author(s):

Kristina B.M. Still ◽

Julien Slagboom ◽

Sarah Kidwai ◽

Chunfang Xie ◽

Bastiaan Eisses ◽

...

Keyword(s):

Fatty Acid ◽

High Resolution ◽

High Throughput ◽

High Throughput Screening ◽

Proinflammatory Mediators ◽

Ph Indicators ◽

Snake Venom Phospholipase ◽

Screening Assays ◽

Animal Venoms ◽

Phospholipase A2 Activity

AbstractMany organisms, ranging from plants to mammals, contain phospholipase A2 enzymes (PLA2s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA2s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA2s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA2s. The methodology was demonstrated for assessment of PLA2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii.

Download Full-text